Endothelial dysfunction contributes to the pathogenesis of atherosclerotic vascular disease in millions of obese, insulin resistant, and type II diabetic patients. Among other risk factors, these patients have elevated levels of circulating free fatty acids. To clarify the mechanisms of endothelial dysfunction in these patients, this proposal focuses on elevated free fatty acids as a diabetes-associated metabolic derangement that contributes to endothelial dysfunction by altering nitric oxide (NO) production. Previous studies, as well as the preliminary data presented in this proposal, clearly demonstrate that fatty acids modulate endothelial nitric oxide synthase (eNOS) activity and NO production in vitro and in vivo. Based on preliminary data, we hypothesize that omega-6 fatty acids cause endothelial dysfunction by inhibiting NO production, whereas omega-3 fatty acids prevent or ameliorate endothelial dysfunction by stimulating NO production. The mechanisms of these alterations in endothelial NO production are postulated to derive from fatty acid-induced differential activation of signaling pathways and peroxisome proliferator-activated receptors (PPAR) which modulate endothelial gene expression. To address these hypotheses, four specific aims will be investigated: 1) define the effect of specific free fatty acids on vascular endothelial NO production in vitro and in vivo, and determine if fatty acids modulate 2) eNOS expression, 3) post-translational modifications of eNOS, or 4) the expression of proteins regulating eNOS expression of proteins regulating eNOS activity through activation of PPARs. After treatment with specific free fatty acids, NO production will be measured in cultured porcine and human aortic endothelial cells in vitro. Similar fatty acids will be infused into rats to determine the effect of specific fatty acids on endothelial- dependent vasorelaxation in vivo. Fatty acid-induced alterations in the expression of eNOS, the activation of protein kinases C and Akt, and the activation of PPARs to modulate the expression of other proteins involved in eNOS regulation will be defined in both in vitro and in vivo models. These novel studies will clarify cellular and molecular mechanisms of endothelial dysfunction that contribute to diabetic macrovascular disease.